Use of Inhibitors of the Renin-Angiotensin System

ABSTRACT

It has been found that inhibitors of the rennin-angiotensin system are useful for the treatment or prevention of conditions associated with hypoxia or impaired metabolic function or efficiency. In particular, they may be used in connection with therapy of stroke or its recurrence, the acute treatment of myocardial infarction, and the treatment or prevention of wasting or cachexia, and are thus useful in treatment of the symptoms and signs of aging. These inhibitors may also be used to enhance function in healthy subjects.

FIELD OF THE INVENTION

This invention relates to the use of inhibitors of the renin-angiotensinsystem.

BACKGROUND OF THE INVENTION

Wasting diseases may be categorised into generalised and localisedwasting diseases. To deal first with generalised wasting, many diseaseprocesses can lead to aggressive generalised weight loss through eitherthe inability to consume sufficient nutrients and energy sources,through their loss from the body (either enterally or in the form ofcellular matter), or through an inability to absorb them. Other diseasesare associated with marked weight loss quite out of proportion to anyreduction in nutrient absorption or increase in nutrient loss. Suchweight loss may have a metabolic origin. Severe cardiac failure as wellas renal, hepatic and malignant disease processes are all associatedwith such inappropriate weight loss. Some neurological diseases, such asParkinson's disease and syndrome are similarly related, as areconditions associated with inflammatory processes, such as severe sepsisor septic shock and autoimmune and connective tissue disorders. Thisweight loss may at best be disabling, and at worst associated with anincreased mortality. Current treatment and preventative strategieslargely focus on nutritional support.

In localised wasting, disuse of any given muscle group (for instance dueto musculoskeletal or neurological injury) may lead to wasting in theaffected territory. There are currently no available treatments whichare routinely used to slow or limit such wasting, nor which have beenshown to accelerate the reversal of such wasting with appropriateexercise or after the cessation of the initiating disease state.

Current strategies for the promotion of trainability and fitness havelargely focused on alterations in training pattern. More recently,nutritional supplementation has been suggested using the manipulation ofscale and nature of intake of carbohydrates, fats, vitamins and aminoacids. The addition of other substrates, such as creatine derivatives,have also been used. Most such interventions are either currentlyunproven, or have been shown to have no or only modest influence.Endocrinological interventions have been attempted, including the use ofandrogens and other steroid hormones. The use of insulin or of growthhormone may also have a role. However, these treatments may beassociated with an unacceptable side-effect profile and also suffer fromthe disadvantage that they have to be parenterally administered (usuallyby intramuscular injection). Pharmacological manipulations are notcurrently available.

The possibility of improving cardiovascular, and other organ, functionis known in connection with the phenomenon of “preconditioning”. Theexposure of an organ—most notably the heart—to a brief period of reducedblood flow or oxygen supply has been shown to provide protection againsta second more severe similar event which might otherwise prove lethal tocells or the organ itself. Much research is currently being undertakenin an effort to identify pharmacological agents which might mimic thisprocess. None is available for routine clinical practice.

The renin-angiotensin system (RAS) and its components may be describedas follows. Briefly, cells of the renal juxta-glomerular apparatusproduce the aspartyl protease renin which acts on the alpha-2 globulinangiotensinogen (synthesised in the liver) to generate angiotensin I(AI). This non-pressor decapeptide is converted to angiotensin II (ATII)by contact with the peptidyldipeptidase angiotensin-converting enzyme(ACE) (reviewed in (1)). ATII stimulates the release of aldosterone, andis also a potent vasoconstrictor. The renin-angiotensin system istherefore important in the maintenance and control of blood pressure aswell as the regulation of salt and water metabolism. Renin,angiotensinogen and ACE have also been identified in cardiovasculartissues including the heart (2) and blood vessels, as has mRNA forcomponents of this system such as angiotensinogen (3-5). Receptors forangiotensin II have been found on vascular smooth muscle cells (6).Within tissues, the RAS may therefore have a local paracrine function(reviewed in (7, 8)), and the expression of the different components canbe altered by pathophysiological stimuli such as sodium restriction (5).Kinetic studies suggest that much of the circulating angiotensin I andII is derived from the both renal and non-renal tissues (9-11).

ACE is a zinc metallo-protease which catalyses conversion of theinactive decapeptide ATI to the active octapeptide ATII thorough thehydrolytic cleavage of dipeptides from the carboxyl terminus His-Leudipeptide. It also catalyses inactivation of bradykinin (a patentvasodilator) by two sequential dipeptide hydrolytic steps; in thiscontext, ACE is also known as kininase II.

The presence of renin-angiotensin system (RAS) components in many animalspecies (such as locusts and elasmobranchs) suggests that they must havesome other role than that of a conventional circulating RAS. Thisfunction must be fundamental and important in order to have beenphylogenetically conserved over many millions of years. In fact,complete renin-angiotensin systems are now thought to exist within manyhuman (and animal) tissues: physiologically-responsive gene expressionof RAS components within these tissues, local generation of ATII, thepresence of ATII receptors and the demonstration that these receptorsare physiologically active have all been shown. Thus, angiotensinogenmessenger RNA (mRNA) is identified in renal, neural and vasculartissues, and local synthesis may strongly influence its concentration ininterstitial fluid (10). Renin mRNA (12) and product (13) is found incultured mammalian vascular smooth muscle cells and throughout thevessel wall (13), and in rat ileum, brain, adrenal, spleen, lung, thymusand ovaries. Liver renin gene expression is physiologically responsive,being increased 3-fold by sodium deprivation or captopril administration(14).

Non-renin angiotensinogenases may also exist in tissues. A neutralaspartyl protease with renin-like activity has been demonstrated incanine brain (15, 16). Some (e.g. tonin, elastase, cathepsin G andtissue plasminogen activator) can cleave ATII directly fromangiotensinogen (16).

ACE expression occurs at high level in vascular endothelium, but also inthe small intestinal epithelium, the epididyrnis (17) and brain (15).Tissue-specific/age-related ACE gene transcription occurs in renaltissue (where there is very high proximal tubular epithelialexpression), and in cardiovascular, hepatic and pulmonary tissues (18).

Such local systems may be paracrine in nature: receptors for ATII areclassically described as existing on cell surfaces, allowingtransduction of the effects of endocrine and paracrine ATII. However,true autocrine systems (intracellular production and actions) may alsoexist. ATII receptors may also exist on the cell nuclei. Specificbinding sites for ATII exist on cellular chromatin which may regulategene transcription (19, 20).

There are many marketed or investigation-stage agents which inhibit RASactivity, and many of them fall into two broad classes: the inhibitorsof angiotensin—converting enzyme, whose approved names generally end in“-pril” or in the case of active metabolites “-prilat”, and antagonistsat angiotensin receptors (more specifically, currently, the AT₁receptor), whose approved names generally end in “-sartan”. Alsopotentially of increasing importance may be a class of drugs known asneutral endopeptidase inhibitors, some of which will also have anACE-inhibitory effect or the potential to reduce RAS activity.

Brink et al. (21) suggested that angiotensin II may have a metaboliceffect in rats (in vivo experimental work) which is independent of itseffects on blood pressure.

There is evidence that angiotensinogen gene expression is differentiallymodulated in fat tissue in obese rats when compared to their equivalentlean strain (22).

ACE inhibition increases rabbit hind leg oxygen consumption at high workloads, but not at lower workloads (23).

ACE inhibitor (ACEI) increases insulin-dependent glucose uptake into theskeletal muscle of an obese rat strain which exhibits relativeinsulin-resistance (24), and this may be kinin-dependent (25). Glucosetransporter levels were elevated in this study,

as they were sustained by AT₁ receptor antagonism in the diabetic ratheart (26).

ATII increases rat hind limb O₂ usage and twitch tension (27). Thispaper concludes that the effects might have been due to effects on bloodflow or neurotransmission and not to a direct metabolic effect.

In heart failure in dogs, fatigue-resistant fibres are conserved by ACEinhibitor therapy (28). In rats, capillary density is maintained, andcollagen volume reduced (29, 30).

Kininases (such as ACE) have been shown to exist in the cell membranesof human skeletal muscle (31). Thus, skeletal muscle RAS may exist (32).

In vitro, ACE inhibitors cause an increase in myocardial oxygenutilisation. Whether this was due to increased or reduced efficiency wasunclear (33). This work related to myocardial muscle extracts. Thiseffect may be due to reduced kinin breakdown, and thus increased kininlevels, despite the fact that angiotensin II may modulate (and increase)kinin release (34).

Other publications suggest an effect of ACE inhibitors or of angiotensinII on muscle performance or metabolism, but all of these have concludedthat the effects are mediated by alterations in nutritive blood flow(35, 36).

In human forearm, kinins increase blood flow and glucose uptake,although again a direct effect of RAS, or an effect on performance, wasnot detailed (37).

Losartan (an AT₁ antagonist) improves insulin sensitivity in humanskeletal muscle (38).

Other publications suggest no beneficial effect of ACE inhibition,amongst those with heart failure in muscle energy balance (39). ACEinhibition did not alter perceived work or maximal work capacity of 20students on a bicycle ergometer (40).

SUMMARY OF THE INVENTION

It has now been found that renin-angiotensin systems are implicated inthe regulation of cellular metabolic efficiency, in the mechanicalefficiency of tissue systems such as cardiac and skeletal muscle, and inthe regulation of growth of cardiac and skeletal muscle. Thisobservation leads to the possibility of down-regulating the activity ofthis system (thus reducing the action of the substance angiotensin IIand increasing the activity of kinins) so as to enhance metabolicefficiency and enhance mechanical performance of tissues. Suchenhancement allows improved management of diseases involving wasting(including severe inflammatory conditions, severe heart failure andmalignant states), the ability to offer relative protection to tissuesfrom periods of reduced oxygen supply and the ability to enhance humanand animal physical performance. In summary, the present invention isbased on the discovery of a previously unknown effect of RAS-inhibitors,i.e. for the promotion of metabolic function or efficiency.

Improvement in metabolic function or efficiency may be seen as:improvement of cellular function and survival in the presence of lowoxygen supply relative to demand; enhancement of mechanical performanceof human skeletal and cardiac muscle; and/or enhancement of nutritionalstatus.

The invention therefore finds application in:

-   -   a. the treatment and prevention of wasting disorders such as        cachexia in malignant disease, acute and chronic sepsis, chronic        hepatic diseases, end-stage renal disease, AIDS and immune        system disorders, and cardiac failure;    -   b. the promotion of cardiovascular fitness, human physical        performance, and physical endurance and the improvement of the        ability of these parameters to respond to physical training, as        well as helping sustain these parameters (this applies to the        physical training of individuals, as well as to the training of        muscles for specifically therapeutic purposes such as        cardiomyoplasty); and/or    -   c. influencing the alteration in body composition and/or        morphology associated with exercise, by altering muscle and fat        content.

There is a need for new methodologies in these areas. This need appliesparticularly to humans, but where appropriate may also apply to thetreatment of other mammals.

In particular, the present invention utilises the availability ofeffective agents with low toxicity and side-effect profiles, and whichmay be administered enterally or parenterally, to allow manipulation ofhuman physical performance. This may fall into four main areas:

-   -   a. sporting applications, including improved sporting prowess        and more rapid recovery of function and performance after        injury;    -   b. military and social situations where enhanced physical        performance may be paramount, e.g. those encountered by military        personnel, fire-fighters and police forces;    -   c. the enhancement of performance in environments where oxygen        supply is diminished, such as at altitude, and in disease states        associated with low tissue oxygen delivery; and    -   d. the enhancement of respiratory muscle training and recovery        of respiratory muscle function after a protracted period of        mechanical ventilation, thus aiding weaning from mechanical        ventilation on intensive care units.

Improved physical fitness would allow improved ability to completetasks. Cardiovascular and cardiorespiratory fitness is also associatedwith reduction in mortality and morbidity rates from cardiovascularcauses.

Agents may be used, in accordance with the invention, to limit tissuedamage (e.g. cerebral or cardiac) in the event of a clinical event ofthe type seen in connection with preconditioning, when applied toindividuals at risk of such events (e.g. those at risk of stroke orheart attack, or those about to undergo a procedure associated with lowoxygen delivery, such as coronary angioplasty or cardio-pulmonarybypass). This effect might also provide protection to cells and tissues,and ultimately to life, in those who are at risk of, or who suffer,exposure to global (rather than tissue-specific) low oxygen delivery.Such individuals would include mountaineers at high altitude (thefunction of whose organs, including the brain and heart, would beimproved, thereby preventing damage to them) and those with severecirculatory failure. Others with severe hypoxaemia who might alsobenefit include those with severe lung disease or circulatoryderangements which are associated with profound hypoxaemia. Suchconditions include infections such as pneumonia, adult respiratorydistress syndrome, pulmonary embolic disease, pulmonary fibrosis,Eisenmenger's syndrome and cardiac left-to-right shunts.

Altering the metabolic efficiency of tissues, as well as the mechanicalefficiency of muscle function (and hence the metabolic demands of thebody), would lead to alterations in body fat utilisation. Further,manipulation of muscle mechanical efficiency may also alter musclegrowth. In this way, improving metabolic efficiency may alter theresponse of body morphology to a period of exercise training and toaltered dietary intake. Such an improvement would also modify a systemwhich may have direct trophic effects on muscle, and might thereforealter skeletal muscle growth by a second mechanism. An improvement inmetabolic efficiency would also limit cardiac growth in response tosevere exercise or pressure burden.

Particular areas of interest, within the context of this invention, arethe treatment or prevention of the effects of ischaemia, includingglobal ischaemia, renal and intestinal ischaemia, stroke, unstableangina, stable angina, myocardial infarction (immediately afteroccurrence), peripheral vascular disease, cerebral palsy, chronic oracute respiratory diseases (which may be associated with hypoxaemia),including respiratory distress syndrome, interstitial lung disease,hypoxaemia, car pulmonale, disorders involving a shunt between thepulmonary and systemic circulations, conditions causing hypoperfusion ofvital organs, cardiac arrest, septic states including meningococcalsepticaemia, sickle cell anaemia, CO poisoning and resuscitation fromdrowning. A use of interest and value is the prevention of hypoxiaduring birth, by administration to the mother, thereby potentiallyreducing the chance of the child being brain-damaged; this isparticularly relevant if it is anticipated that the birth will bedifficult.

Evidence presented below indicates that the effect of RAS inhibitors onmitchondrial function is consistent with the theory presented herein, Italso explains the utility of such agents in cardiac problems, butbroadens the scope of their utility, e.g. to non-cardiac uses, in brain,liver, kidney etc, and in skeletal muscle; Cells are able to functioneffectively under conditions of reduced oxygen availability, and/or toutilise oxygen more efficiently. Thus, in connection with the treatmentor prevention of stroke or its recurrence, the penumbra ofoxygen-starved cells around a clot or hemorrhage can function moreefficiently. The stroke may be thrombotic or hemorrhagic,cerebrovascular or accident in origin. Further, a RAS-inhibitor may beof benefit in the transport or survival of transplanted organs.

The invention has utility in therapy in general, in the treatment andalso the prevention of adverse conditions. It has utility in treatingsymptoms associated with such conditions, e.g. wasting. It also hasutility in enhancing performance where the subject would normally beregarded as healthy, i.e. without reference to any particular adversecondition. One particular area of interest is ageing, i.e. where thesubject may or may not be ill, but where use of the invention canpositively affect the well-being of the subject,

Inhibitors of RAS have been given to subjects having raised bloodpressure, and it may be that this will have provided effects associatedwith the present invention. An aspect of the present invention is therealisation that such agents are useful when the subject has normalblood pressure, and that the effects are independent of any effect onblood pressure. The invention is of value where undue reduction in bloodpressure does not occur, or is not a problem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the pre-treatment of cardiomycocytes with 1 μM lisinoprilfor 36 hours caused an increase in Rh123 fluorescence of about 30%,indicating that ACE inhibition induced an increase in ΔΨ_(m).

FIG. 2 shows that treatment of rat cardiomyocytes with 1 μM lisinoprilfor various lengths of time caused a progressive increase in redfluorescence () with a corresponding decrease in green fluorescence(◯). Thus, the ratio of red to green fluorescence (□) increased as thetime of incubation with lisinopril progressed.

FIG. 3 shows angiotensin II plasma levels for a variety of cachecticconditions.

DESCRIPTION OF THE INVENTION

The invention may be utilised to affect any RAS. Amongst other tissues,local tissue renin-angiotensin systems have been suggested in the brain,blood vessel wall, heart, intestine, liver and kidney.

Having described the various components of the RAS above, it will beapparent that the system can be inhibited at various points. Inprinciple, it is expected that any sufficiently non-toxic compound whichis bioavailable and active to inhibit the RAS system at any suitablepoint can be used in the invention. This invention contemplates theadministration of all such agents (either singly or in combination witheach other and/or with other classes of pharmacological agents), andalso of pro-drugs which are converted in vivo to an active agent whichinhibits RAS activity. Note that RAS inhibition need not be totalinhibition; rather, sufficient inhibition to be beneficial in theinvention is all that is required. In practice, it is preferred at thepresent state of knowledge to use in the practice of the invention anyof the known RAS inhibitors which are either on the market or underinvestigation for their antihypertensive effects.

Many inhibitors of the renin-angiotensin system are licensed or underinvestigation for use in humans in the United Kingdom and are compoundswhose use is preferred in the practice of the invention. They includethe ACE-inhibitors Quinapril, Captopril, Lisinopril, Perindopril,Trandolapril, Enalapril, Moexipril, Fosinopril, Ramipril, Cilazapril,Imidapril, Spirapril, Temocapril, Benazepril, Alacepril, Ceronapril,Cilazapril, Deiapril, Enalaprilat and Moveltipril. Suitable angiotensinII-inhibitors include Losartan, Valsartan, Irbesartan, Candesartan,Eprosartan, Tasosartan and Telmisartan.

The specific compounds listed may be useful in accordance with theinvention in their free form, for example as the free acid or base asthe case may be, and they may be useful as acid addition salts, esters,N-oxides or other derivatives as appropriate. The use of suitablepro-drugs (whether themselves active or inactive) and the use of activemetabolites of RAS inhibitors are also within the scope of theinvention. For example, alacepril is a pro-drug for captopril, andenalaprilat is an active metabolite of enalapril.

Although ACE inhibitors and angiotensin II-receptor antagonists arepresently the most widely developed classes of drugs suitable for use inthe present invention, the invention is by no means limited to theiruse. Other inhibitors of the RAS system include resin inhibitors andneutral endopeptidase inhibitors: ACE inhibitors may work through both areduction in ATII formation and through a reduction in kinin metabolism.Other agents may also inhibit kinin degradation, and as such havesimilarly beneficial effects. These classes of drugs include inhibitorsof neutral endopeptidases, some of which also of ACE-inhibitoryproperties. The invention thus contemplates the use of allkininaseinhibitors and kinin receptor antagonists (such as bradykinin).

In many circumstances, it may be that a combination of thetissue/metabolic effects of such antagonists to the RAS with theirsystemic effects (e.g. reduced blood pressure, reduced cardiac preloador afterload and vasodilatation) and other combined effects (e.g.ventricular remodelling) may be of value. Such circumstances might be inthe treatment of patients with hypertension, peripheral vasculardisease, cardiac failure or cardiac hypertrophy.

In normotensive subjects, or in hypotensive individuals (either throughthe effect of other drugs, through natural phenotype, or through diseasestates such as sepsis or septic shock) any further reduction in bloodpressure or other systemic effects of RAS antagonists might bedisadvantageous. Under such circumstances, the use of lipophilic, oreven highly lip ophilic, agents may have advantages in enablingtissue-RAS inhibition to be achieved without effect on systemic bloodpressure. That this can be done in animals has been shown by manygroups. Indeed, even in a profoundly hypertensive animal model, 5μg/kg/day of ramipril administered to rats had no effect on systolicblood pressure. This technique of using very low doses of a lipophilicACE inhibitor has also been applied to humans: a low dose of ramiprilcould produce significant biological effect without any recordableeffect on systemic blood pressure (41).

The invention contemplates the use of compounds which are essentiallynon-lipophilic, or only moderately lipophilic, but which have beenrendered more lipophilic either chemically, such as by appropriatederivatisation, or physically, such as by formulation with lipophiliccarriers or delivery systems.

Compounds having activities as described above are useful, in accordancewith the invention, for promoting metabolic function or efficiency andhence improved biochemical and mechanical function. This may be achievedthrough a variety of mechanisms (above) which may include:

-   -   improved blood supply (and hence substrate supply);    -   increased substrate uptake (e.g. of glucose or oxygen); and/or    -   improved cellular efficiency in the use of these substrates        (e.g. achieving the same mechanical or biochemical work for the        use of less oxygen or metabolic substrates).

The first two examples may be regarded as improved metabolic function,and the third may be regarded as improved metabolic efficiency.

In particular, it is envisaged that the invention will be useful intreating those conditions, and addressing those situations, in relationto which it was discussed above that there was currently an unmet need.These include treating wasting diseases, promoting trainability andfitness, and altering body composition and/or morphology. Generallyspeaking, a RAS inhibitor may be administered at any effective buttolerated dose, and the optimum dose and regimen can be establishedwithout undue difficulty by essentially conventional trial work. Somegeneral guidance follows, but ultimately the appropriate dosage andregimen of each drug for the various conditions within the ambit of theinvention will be within the control of the clinician or physician. Ingeneral, compounds useful in the invention may be given by oral therapy(by mouth) or enteric therapy (administration through nasogastric,nasoenteric or other enteric feeding tubes) or parenterally, such asintravenously, for example by the addition of compound(s) to bags ofparenteral nutrition.

Generalised wasting: It has been discussed that many disease processes,including severe cardiac, renal, hepatic and malignant disease,respiratory disease, AIDS, and chronic or acute inflammatory processessuch as severe sepsis (or septic shock) and autoimmune and connectivetissue disorders, can lead to a generalised weight loss through ametabolic mechanism. The present invention enables the prevention ortreatment of such conditions with the RAS inhibiting agents as describedabove. It is anticipated that low doses of such agents (e.g. 1.25 mg oframipril) may be effective. In principle, however, a similar strategy tothat used in the treatment of heart failure would seem most likely to beused, namely a steady increase in dosage to a maximum tolerated. Themajor limiting factors in treatment may be:

-   -   a. The development of cough in some individuals treated with an        ACE inhibitor, although switch to another agent or class of        agent might be possible; and/or    -   b. A significant fall in blood pressure. At doses of 2.5 mg        ramipril (or equivalent of other agents), a first-dose fall in        blood pressure occurs with the same frequency as is seen with        placebo in trials of treatment of acute myocardial infarction,        suggesting that in many this sort of dose would be safe.

Appropriate doses for critically-vasodilated patients (such as thosewith septic shock) would be established following appropriate protocolsknown to those skilled in the art and/or by titration to an individualpatient.

Localised wasting: Dosage of the RAS inhibitor may be at the maximumtolerated dose, as in the published range for each agent for use intreating heart failure or hypertension. Low doses (such as 1.25 mgramipril) may allow benefit without any significant hypotensive effect,as discussed above.

Preconditioning: A suitable preventative strategy would involve givingthose at risk of

organ ischaemia (e.g. those with significant risk of myocardialinfarction or stroke) a regular dose of the agents described. Those withknown poor cardiac, skeletal muscle (e.g. claudicants) or cerebral flowmight also benefit from treatment, through enhancing metabolicefficiency, and providing cellular protection to critically-ischaemiccells until such time as revascularisation might be considered. Dosageof the agent classes at the maximum tolerated dose, as in the publishedrange for each agent for use in treating heart failure or hypertension.Low doses (such as 1.25 mg ramipril) may allow benefit without anysignificant hypotensive effect, as discussed above. It may be possibleto use parenteral formulations to provide protection to those who havejust suffered such an ischaemic event or to those about to undergo aprocedure leading to ischaemia, such as angioplasty or cardiac bypass.

Promotion of trainability and fitness: Dosage of a RAS inhibitor may begiven at the maximum tolerated dose, as in the published range for eachagent for use in treating heart failure or hypertension. Low doses (suchas 1.25 mg ramipril) may allow benefit without any significanthypotensive effect, as discussed above. Administration of a RASinhibitor to those with peripheral vascular disease might be expected toimprove exercise endurance and possibly limb viability through acombination of the mechanisms contemplated herein.

Alteration in body composition and/or morphology: Dosage of a RASinhibitor may be given at the maximum tolerated dose, as in thepublished range for each agent for use in treating heart failure orhypertension. Low doses (such as 1.25 mg ramipril) may allow benefitwithout any significant hypotensive effect, as discussed above.

As far as formulation and administration are concerned, it is expectedthat the various drugs useful in the invention could be administered inthe same formulations as currently exist. New formulations might bedeveloped with the express intent of being

able to exert a predominantly tissue-effect without significant systemichypotensive effects, in the same way as has been described for low-doseramipril, or for local tissue delivery or for intravenous orintra-arterial administration. Currently, there has been an emphasis onthe oral administration of most of these agents. However, formulationsto allow systemic parenteral administration may enhance the ability totreat the critically ill, or those undergoing interventions leading tovascular occlusion or low blood flow rates as indicated above.Additionally, new formulations (for example, for local delivery, asalready mentioned) may become available.

Administration of the active agent may be by any suitable route. As isconventional for ACE inhibitors at least oral administration may bepreferred, especially for the purposes of achieving a prophylactic orpreventative effect. In certain circumstances, especially when a moreimmediate effect is required, intravenous administration may bepreferred; for example, a subject who has just experienced an infarctionmay be given the active agent intravenously, not for the purpose ofremodelling but to alleviate local oxygen demand and thereby facilitatetreatment. Suitable formulations for intravenous administration will beevident to those skilled in the art.

In the above discussion, indicative doses have been given, by way ofexample only, as optimal doses may be established experimentally and/orclinically. It should be noted that useful doses in accordance with theinvention may be below optimal anti- hypertensive doses or even beloweffective anti-hypertensive doses.

The optimum frequency of dosage and duration of treatment may also be

established experimentally and/or clinically. Again by way of example,oral ramipril may be given once daily for an appropriate period of time.Frequencies of dosage for other compounds useful in the invention willvary, and will depend on, among other things, the pharmacokinetics ofthe compound in question.

The invention enables the provision of a method of promoting metabolicfunction

or efficiency, the method comprising administering to a subject aninhibitor of the renin-angiotensin system. The inhibitor will generallybe administered in an amount which is non-toxic or only acceptably toxicbut which is effective to promote metabolic function or efficiency (orof course both). The subject will generally be human, but non-humananimals may also benefit from the invention. Promotion of metabolicfunction or efficiency may be undertaken, for example, for therapeutic,prophylactic, social, military, recreational or other purposes.Preferred features of such a method of treatment are as described above.

Other aspects of the invention include:

a method, which may be a non-therapeutic method, of promoting metabolictrainability or fitness in a healthy subject, the method comprisingadministering to the subject an inhibitor of the reninangiotensinsystem; and

a method, which also may be a non-therapeutic method, of altering bodycomposition and/or morphology in a healthy subject, the methodcomprising administering to the subject an inhibitor of thereninangiotensin system.

By way of illustration of circumstances in which this invention may beused, mountaineers may take ramipril at low dose (1.25-2.5 mg) for 4weeks prior to their departure whilst training, so as to improve theirtrainability, recognise any side effects prior to departure, and to loadtheir tissues with the drug. They continue to take the drug whilst ontheir expedition.

Another example is of an elderly, injured patient on a ventilator, whomay be given a test dose of a short-acting ACE inhibitor (captopril),and side-effects (such as a decline in renal function or fall in bloodpressure) watched for. The dose of ACE-inhibitor (given nasogastrically)is then increased to a maximal tolerated dose (such as 20 mg bd orenalapril, or 10 mg od ramipril). It is intended that this interventionwill slow the anticipated muscle wasting (both generalised systemic, andlocal wasting from disuse atrophy).

A patient may be treated post-surgery, in the same way. Respiratorymuscles are ‘trained’ by steady reduction in mechanical ventilatorysupport, and this training is enhanced by the therapeutic use of the ACEinhibitor.

A similar regime may be suitable for the treatment of a patient with lowsystemic oxygen levels due to severe lung injury from Adult RespiratoryDistress Syndrome associated with a systemic inflammatory responsesyndrome, or severe smoking-related lung disease. It may also be usedfor a patient due to undergo coronary angioplasty, e.g. ramipril for 4weeks prior to the procedure, for a patient who has suffered a coronaryocclusion, and is considered at risk of further events, or who hasperipheral vascular disease. Further, it may be used to manage a patientwho has suffered a femoral arterial occlusion and undergone angiography;an infusion of thrombolytic agent is administered into the femoralartery directly, along with an intraarterial ACE inhibitor at the samesite, to improve muscle survivability as reperfusion occurs. Anotherexample of a suitable subject for treatment is overweight, and who findsit hard to exercise and lose weight; an ACE inhibitor may be given, inassociation with an intensive exercise training programme. Yet anothersuitable patient has smoked heavily throughout his life and suffersintermittent claudication at a distance of only 150 yards.

The invention may also be used in the treatment of subjects exhibitingsevere cachexia, as has been observed in cases of TB, HIV, pleuraleffusion, meningitis, hepatitis, perferated stomach ulcer, livercirrhosis, cellulitis, hepatoma, sickle cell anemia, appendicitis,sinusitis, dysphagia, abcess, pneumonia, chronic diarrhoea,encephalopathy and bone fracture.

As will also be apparent from the present disclosure, the inventionincludes within its scope various screening methods, including a methodof diagnosing or screening an individual for an inherited predispositionto promotability of metabolic function or efficiency in a subject, themethod comprising analysing detecting in the individual an allele of theACE gene (DCP1) on chromosome 17q23.

The invention therefore encompasses each of:

A method of screening an individual for response to treatment orprevention of wasting;

A method of screening an individual for response to cardiacpreconditioning;

A method of screening an individual for response to promotingtrainability and fitness; and

A method of screening an individual for response to altering bodycomposition and/or morphology;

wherein, in each case, the method comprising analysing detecting in theindividual an allele of the ACE gene (DCP1) on chromosome 17q23.

In particular, such methods may comprise determining the presence(Insertion, I) or absence (Deletion, D) of a 287 base pair alu repeatsequence in intron 16. The methods may be carried out in vitro ifappropriate.

ACE gene I/D genotype may be determined in a number of ways. Mostcurrently rely upon polymerase chain reaction amplification of genomicDNA which may be derived from a number of sources. Most commonly, bloodor mouthwash are used. Different primers allow specific amplification ofthe D of I alleles, and the corresponding fragments are then separated,usually by electrophoresis. One example of a suitable technique isdisclosed in Montgomery et al., Circulation 96(3) 741-747 (1997),published 5 Aug. 1997.

By way of further elaboration, the 1/D polymorphism may be identified bypolymerase chain reaction amplification (PCR) and subsequentelectrophoretic separation of fragments. Two PCR methods in particularmay be used. The first-reported method of PCR amplification used twoprimers, has since been used in the majority of the published studies,e.g. as described by Cambien et al, Nature 359 641-644 (1992). It hassince become clear that this system is prone to systematic bias in thatthe shorter (deletion) fragment is preferentially amplified at theexpense of the larger insertion (I) allele. This causesmisclassification of a small proportion (5-15%) of heterozygotes asbeing D homozygotes (Shanmugam et al, PCR Methods Appl. 3 120-121(1993)). Such misclassification may be prevented by specific alterationsin the PCR conditions (such as the addition of a denaturing agent suchas desmethylsulphoxide, which increases the stringency of the reaction),or by the use of an insertion allele-specific third primer as describedby Evans (Evans et al, Q. J. Med 87 211-214 (1994)).

A 3-primer PCR system, with primers as described by Evans (Evans et al,loc cit, 1994) may have a modified protocol as subsequently described:Two priming oligonucleotides flank the insertion sequence in intron 16and a third oligonucleotide is specifically within the insertionsequence. This method yields shorter allele fragments. This, togetherwith I-allele-specific amplification, eliminates the mistyping ofheterozygotes as DD homozygotes. We used primer ratios corresponding tothe 50 pmol ACE1 (5′ or left hand oligo) and 3 (3′ or right hand oligo)and 15 pmol ACE2 (insertion specific oligo) used by Evans et al in a 50μl reaction, giving amplification products of 84 bp for allele ACE D and65 bp for allele ACE I. Our amplification conditions were as follows: 1cycle 95° C. 5 min; 40 cycles 95° C. 1 min, 50° C. 1 min, 72° C. 5 min,20 μl PCR reactions contained 50 mM KCI, 10 mM Tris HCl pH 8.3, 1.5 mMMgCl₂, 0.01 mg/ml gelatin, 200 μM each dNTP, 0.2 units Taq polymerase(Gibco BRL, Paisley, UK) and 8 pmol of primers ACE1 and ACE3, outsidethe insertion (Alu) sequence, and 2.4 pmol of primer ACE2, inside theinsertion sequence. Reactions were overlaid with 20111 mineral oil. All96 wells were always filled with reagents (mix or dummy reagents) toensure constant thermal mass on the block. Amplification products werevisualised using electrophoresis on 7.5% polyacrylamide gels. Theaccuracy of our genotyping was confirmed under conditions previouslyreported (O'Dell, Humphries et al, Br. Heart 73 368-371 (1995)), suchthat replica PCRs set up using only the primer pair ACE I and ACE3, bothat 8 pmol per 20 μl PCR reaction, always confirmed the presence of the Dallele.

DNA fragments were separated using agarose gel electrophoresis (in thecase of the 2-primer system), and electrophoresis on an 8.4%polyacrylamide gel (in the case of the 3-primer system). Fragments wereidentified by the incorporation of ethidium bromide into the gels, andviewing under ultraviolet light.

Preferred features of each aspect of the invention are as for each otheraspect mutatis mutandis.

Large interindividual differences in plasma ACE levels exist, but levelsare similar within families (42), suggesting a strong genetic influencein the control of ACE levels. The human ACE gene (DCP1) is found onchromosome 17q23 (43) and contains a restriction fragment lengthpolymorphism (44) consisting of the presence (Insertion, I) or absence(Deletion, D) of a 287 base pair alu repeat sequence (45) in intron 16(46). D allele frequency is approximately 0.57-0.59 (43, 45).

This I/D polymorphism has been shown to influence circulating ACElevels. Amongst 80 healthy Caucasians, the polymorphism accounted for47% of the variance in plasma ACE, although considerable overlap existedbetween groups (44). Tissue ACE levels might be similarly influenced.T-cells express ACE. The facts that most of the ACE activity ismicrosomal, and that B-cells lack ACE mRNA expression while monocyte ACElevels are 28-fold lower, support the conclusion that T-cell ACEactivity is due to cellular synthesis not passive adsorption from thecirculation. ACE activity in those of DD genotype is 75% and 39% higherin plasma and T-lymphocytes, respectively, than in those of II genotype(47). Local ATII generation in human internal mammary artery may also beincreased in those of DD genotype (48). Cardiac ACE activity may besimilarly influenced (49). However, there is no evidence of anassociation of ACE genotype with circulating ATIL levels (50). Thesedata suggest an influence of the I/D polymorphism on tissue and plasmaACE activity. Increasing D-allele burden might thus be associated withincreased ‘net RAS activity’ in tissue systems. Any phenotypecritically-regulated by tissue RAS may be more prominent within apopulation amongst those of DD genotype if tissue ACE levels are therate limiting step in the tissue RAS. Many physiological stimuli causeinduction of RAS (including ACE) gene expression. Prospective studies ofpolymorphism influence on the phenotypic response to a physiologicalchallenge therefore allow not only elucidation of a role for tissue RASin the control of that phenotype, but also examination of the molecularcontrol of tissue ACE expression. For instance, if the D allele isassociated with more responsive gene transcription, any givenphysiological challenge will cause a disproportionate change inRAS-dependent phenotype in association with the D allele.

The present invention is not restricted to administration of activeagents to individuals of a particular genotype. However, it is evidentthat the benefits of the invention may be seen in circumstances wherethere may be elevated levels of, say, ACE. This lends support to theinvention. A study was conducted, in which various parameters weremeasured, at the start and end of a 10 week physical training period inmale Caucasian military recruits. A possible influence of ACE genotypeon systolic blood pressure is seen in the cohort as a whole, i.e.amongst the individuals who completed training. This trend is notstatistically significant prior to training (p for heterogeneity=0.35),but approaches significance at the end of training (p forheterogeneity=0.07) when systolic blood pressure for those of IIgenotype was significantly lower than those of DD genotype (122.7±1.4vs. 118.0±1.5 mmHg: p<0.05). Diastolic blood pressures did not differbefore or after basic training between those of different genotype(pre-training 70.3±1.37 vs. 70.6±0.8 vs. 69.4±1.3 mmHg, p=0.75:post-training 69.7±1.23 vs. 70.1±0.81 vs. 69.9±1.23 mmHg, p=0.96: forII, ID and DD respectively).

In another study, the upper limb performance of army recruits wasobserved, since they are specifically trained for power during armybasic training. Tests were conducted at selected timepoints, i.e. thestart of training, mid-training (5 weeks), and end-training (10 weeks).

For paired data on calf strength, there was a suggestion of a genotypeeffect. Mean of paired percentage changes were 7.6±14.1 vs. −6.4±4.1:p=0.19.

At the start of training, there were no differences in biceps power(109.4 N±8.5 vs. 111.5 N±5.6 for II vs. D-allele: p=0.86). However, atthe end of training, there had been a significant improvement in bothgroups, but to a much greater degree amongst those of II genotype(198.7±26.1 vs. 141±9.37 for II vs. D-allele: p−−3.01). The meanpercentage change was 77.0±24.4% vs. 23.7±6.2% for II vs. D-allelerespectively: p=0.003). Mean changes for those of II genotype were 109.4vs. 198.7, compared to 109.8 vs. 144.9 for those of ID genotype, and116.4 vs. 125.9 for those of DD genotype: II<ID, and II<DD with p<0.05at end of training). The data are in duration of exercise (seconds).

Data for press-ups were similar at start of training (mean 51.2±4.0 vs.50.4±2.4 vs. 47.5±4.3: n=29, 69 and 22: for II, ID and DD respectively:p>0.05 for all comparisons. At the end of training, the figures were61.9±4.2 vs. 59.5±3.6 vs. 45.0±6.2: n=19, 44 and 12: for II, ID and DDrespectively: p<0.05 for II vs. DD and ID vs. 20 DD).

The change in VO₂max (from a baseline level similar across genotypes)was +0.055±0.037 vs. −0.003±0.021 vs. −0.068±0.052: p<0.05 for II vs.DD). [V0₂max is the maximal oxygen consumption (in ml) per unit time(min.)]

Weight increased significantly more for those of II genotype than thosewith a D allele (by 2.9±0.8% vs. −0.1±0.6%: n=20 vs. 61: p=0.01: p<0.05for II vs. ID). This is a balance of changes in fat and muscle, both ofwhich might be differentially regulated by the ACE genotype.

Percentage body fat was similar at the outset (8.6±0.7% vs. 9.3±0.3%:n=29 vs. 93 for II vs. D allele: p=0.33). However, this changed by afraction of 0.20±0.09 vs. 0.007±0.003: n=20 vs. 58: p=0.02, i.e. IIfractional fat content increased by about 20% vs. less than 1% for thosewith a D allele.

Those with a D allele had a slightly lower lean body mass at outset(64.6±1.2 vs. 62.5±0.67: n=29 vs. 93: p=0.12), but this gap widenedafter training (65.9±1.3 vs. 62.6±0.82: n=19 vs. 57: p=0.046).

Fat mass was similar at outset (6.18 kg±0.54 vs. 6,49 kg±0.26: n=29 vs.93: p=0.57). Change in fat weight was genotype-dependent (0.73 kg±0.39vs. −0.26 kg±0.20: n=19 vs. 57: p=0.02), i.e. mean of percentage changesin body fat mass were an increase of 23% for those of II genotype vs. achange of just 1% amongst those with a D allele.

In another study, 33 elite unrelated male British mountaineers with ahistory of ascents beyond 7000m without the use of supplemental inspiredoxygen were identified by the British Mountaineering Council. DNA wasextracted from a mouthwash sample of the 25 male respondents, and ACEgenotype determined using a three-primer polymerase chain reaction (PCR)amplification (51). Genotype distribution was compared to that of 1906British males free from clinical cardiovascular disease (52). Mean (SD)age was 40.6 (6.5) years in the 25 subjects, and 55.6 (3.2) yearsamongst the 1906 controls. Both groups were in Hardy Weinberg

Equilibrium. Both genotype distribution and allele frequency differedsignificantly between climbers and controls (p 0.02 and 0.003respectively), with a relative excess of II genotype and deficiency ofDD genotype. Amongst the 15 climbers who had ascended beyond 8000mwithout oxygen, none was of DD genotype [6 (40%) II and 9 (60%) ID: Iallele frequency 0.65]. Further, ranked by number of ascents withoutoxygen, the top performer climbing over 8000m was of II genotype (5ascents, compared to a mean of 2.4+0.3 ascents for the >8000m group, or1.44+0.3 ascents for the climbers overall), as were the top two in thisgroup for number of additional 7000m ascents (>100 and 18, compared to amean of 10.3+6.5 ascents).

Further, among athletes, an excess of the I allele is found amongstendurance runners, and an excess of the D allele amongst sprinters.Provisional data suggest that the D allele is found in excess inathletes in whose sport power (rather than endurance) plays an importantrole.

These data suggest that many aspects of human physical performance maybe associated with the I allele, and thus with lower tissue ACE levels.Thus, total cardiac work is higher per unit of external work amongstthose with two D alleles than those without, and ability to train toimprove calf strength, biceps power, and press-ups were all associatedwith the I allele, with trainability being graded as II>ID>DD. Thesechanges in performance may be partly related to changes in bodycomposition, with a preservation of body mass and slight overallanabolic effect being associated with the I allele when compared to alack of anabolism (or slight catabolic effect) being seen in those witha D allele. The marked changes in performance by genotype with moremodest changes in muscle mass suggest that there is not only agenotype-associated effect on performance mediated through muscle bulkper se, but also an effect mediated through efficiency of musclemetabolism. This hypothesis is supported by the genotype-effect onenergy stores in the form of fat.

Since the I allele is a surrogate marker for lower tissue ACE levels, itwould seem likely that increased skeletal muscle performance, metabolicperformance, limitation of catabolism, and promotion of anabolism mayall be achieved by reducing tissue RAS activity pharmacologically. Boththe inhibition of kinin degradation and antagonists to receptors forATII might be expected to have such effects. The above data thereforesuggest a metabolic role for human renin-angiotensin systems which hassignificant effects on the human as a whole.

In hindsight, although there are data in the prior art to supportpossible beneficial effects on muscle blood flow and glucose uptake indiseased states, there are no data to suggest any clinically orphysiologically significant effects on whole body morphology, muscle orwhole human physical performance, or on overall nutritive ormorphological state. The data, however, do provide support and potentialscientific rationale for the present invention.

These data suggest that endurance performance may indeed be improved bytreatment with the specified agents. Pure power performance might alsobe improved, but possibly less effectively. The effects on mixed sportmight depend very much on the relative contributions of power andendurance to success.

There might be a number of means through which the observed andanticipated effects might be mediated. These include:

-   -   (i) an increase in blood flow to tissues through vasodilation;    -   (ii) an increase in blood flow to tissues through angiogenesis        (the growth of new vessels);    -   (iii) subsequent on (i), a fall in peripheral vascular        resistance and an increase in cardiac output;    -   (iv) an increase in metabolic fuel (oxygen, fats, carbohydrates,        and amino acids) uptake by tissues;    -   (v) an alteration in the balance of the fuel utilised (such as,        for example, a shift towards the use of fatty acids from which        more energy can be derived than from equivalent amounts of        glucose);    -   (vi) an alteration in the supply of fuel from, for example, fat        and liver stores;    -   (vii) a primary shift in both qualitative and quantitative        substrate metabolism (such as lactate metabolism) and energy        store release (such as fatty acid release) by metabolically        active tissues including the liver;    -   (viii) a change in skeletal muscle cell type, reflected perhaps        in a change in the relative numbers of type I and type II        myocytes. This may be an important factor in the changes in        performance which we are seeing.    -   (ix) a change in the numbers of mitochondria within cells;    -   (x) a change in the efficiency of metabolism within a cell or        organism, reflected by the ability to perform more external,        mechanical, or biochemical work for a reduced utilisation of        oxygen or metabolic substrate or energy.

Other mechanisms may also apply.

The following Examples illustrate the invention and the evidence onwhich it is based, and also show how it may be put into effect inparticular instances.

EXAMPLE 1

This Example demonstrates that ACE inhibitors increase the mitochondrialmembrane potential of cardiomyocytes. It is based on observation of thepotential difference (ΔΨ_(m)) across the inner mitochondrial membranethat is generated by the extrusion of protons to the outside of themitochondrion during the transport of electrons from electron-carryingcoenzymes to molecular oxygen. Part of the energy stored in ΔΨ_(m) isutilised to support the synthesis of most of the ATP derived fromaerobic metabolism. Thus, ΔΨ_(m) is an indicator of the energisationstate of the mitochondrion, and also of the efficiency of oxygenutilisation to generate chemical energy. To investigate whether some ofthe therapeutic properties of ACE inhibitors could be accounted for byan increase in ΔΨ_(m), this parameter was examined in ratcardiomyocytes, following pre-treatment with the ACE inhibitorlisinopril.

More particularly, cardiomyocytes were isolated from new-bornSprague-Dawley rats hearts and maintained in 30 mm tissue culture dishesin the presence of DMEM supplemented with 1% foetal calf serum at 37° C.in a humidified 5% CO₂ atmosphere. For experiments, cultures weretreated with 1 μM lisinopril or with an equivalent amount of vehicle forvarious lengths of time, before analysis of ΔΨ_(m).

To measure ΔΨ_(m), the mitochondrial-specific probes rhodamine 123(Rh123) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanineiodide (JC-1) were used. Cells were incubated for 15 min with 2.5 μMRh123 or for 10 min with 10 μ/ml JC-1 (Molecular Probes) in freshculture medium, at 37° C. and 5% CO₂. The cells were then washed twicewith cold PBS, resuspended by trypsinisation and stored in the dark at4° C. until the time of analysis (usually within 30 min). Flow cytometrywas performed on a FACScan instrument. Data were acquired and analysedusing Lysis II software (Becton Dickinson).

Results:

Cationic lipophilic fluorochromes such as Rh123 serve as reportermolecules to monitor mitochondria] activity. These dyes accumulate inthe mitochondria’ matrix in accordance with the Nernst equation. Whenused in combination with flow cytometry, they are effective probes toestimate changes of ΔΨ_(m) in intact cells. As shown in FIG. 1,pre-treatment of cardiomyocytes with 1 μM lisinopril for 36 hours causedan increase in Rh123 fluorescence of about 30%, indicating that ACEinhibition induced an increase in ΔΨ_(m).

JC-1 is a more reliable and sensitive fluorescent probe for assessingchanges in ΔΨ_(m). At low concentrations, JC-1 exists mainly in amonomeric form which is characterised by the emission of greenfluorescence. Upon accumulation in the mitochondrial matrix JC-1 formsJ-aggregates in proportion to the magnitude of ΔΨ_(m). These aggregatesare characterized by the emission of red fluorescence. Thus, an increasein the red to green fluorescence ratio indicates an increase in ΔΨ_(m).FIG. 2 shows that treatment of rat cardiomyocytes with 1 μM lisinoprilfor various lengths of time caused a progressive increase in redfluorescence () with a corresponding decrease in green fluorescence(◯). Thus, the ratio of red to green fluorescence (□) increased as thetime of incubation with lisinopril progressed.

These experiments demonstrate that treatment with ACE inhibitorsincreases ΔΨ_(m). This indicates that ACE inhibitors may protect againstischaemic situations and/or improve mechanical/biosynthetic performanceby increasing the efficiency of energy transduction in themitochondrion.

EXAMPLE 2

Ninety military recruits were studied before and after military trainingof 12 weeks duration. These were randomised to receive the AT1-receptorantagonist Losartan or placebo.

There was a consistent trend for the recruits to improve their VO2max atanaerobic threshold, although a distinction was observed, according togenotype. The results shows a gain of 2.1±6.8 ml/min for II genotype onplacebo vs. −1.1±6.5 ml/min for DD genotype on placebo, and a gain of0.3±6.3 ml/min for II on losartan vs. −1.8±6.3 ml/min for DD onLosartan. When combined, the difference in gain was 1.3±6.6 ml/min forII on vs. −1.4±6.4 ml/min for DD: p 0.07).

The data for VO2max showed a similar trend, as did measures of musclefatigue. These data are consistent with an enhanced ability, especiallyfor those of II genotype (and thus lower ACE activity) to achieve higherworkloads, before reaching anaerobic threshold, and therefore to be moreresistant to fatigue in situations of moderate to intense exercise.

EXAMPLE 3

The bioactive element ofthe resin angiotensin system (RAS) isangiotensin II (ATII). Elevations of AT II in plasma or in local tissuewould indicate conditions in which inhibition of the RAS may havesignificant therapeutic benefit even where partial inhibition of the RAShas been achieved (such as by therapy with ACE inhibitors).

ATT II was measured as follows: Blood samples were collected aftersupine rest of at least 10 minutes. An antecubital polyethylene catheterwas inserted and 10 ml of venous blood were drawn. After immediatecentrifugation, aliquots (EDTA plasma sample) were stored at −70° C.until analysis. Angiotensin II was measured using a commerciallyavailable radioimmunoassay (IBL, Hamburg, Germany, sensitivity 1.5pg/ml). After extraction of the plasma samples, AT II is assayed by acompetitive radioimmunoassay. This radioimmunoassay is using a rabbitanti-AT II antiserum and a radio-iodinated AT II tracer. Bound and freephases are separated by a second antibody bound to solid phaseparticles, followed by a centrifugation step. The radioactivity in thebound fractions is measured and a typical standard curve can begenerated. The test has a cross-reactivity with AT 1 of <0.1% and awithin and between run reproducibility between 3.9 and 8.6%. Thereference range for healthy subjects is 20 to 40 pg/ml.

A variety of cachectic conditions, for instance due to chronic heartfailure, AIDS, liver cirrhosis, and cancer has been studied. Results arepresented in FIG. 3, where the bars (from left to right) relate to AIDScachexia (n=6), cancer cachexia (n=7), cardiac cachexia (n=17),idiopathic cachexia (n=2), liver cirrhosis cachexia (n=6), malnutrition(n=6) and non-cachectic heart failure (n=11).

Activation of the RAS has been found, in the cachectic conditions, asevidenced by elevated plasma AT II levels (mean AT II plasma levels wereclearly above the upper limit of the normal range of 20 to 40 pg/ml).This is not dependent on any specific aetiology for the cachecticdisorder; in fact, elevated AT II plasma levels (i.e. RAS activity) arealso found in cases of idiopathic cachexia, i.e. cachexia of unknownorigin. Nevertheless, activation of the RAS is apparently specific forcachectic disorders, as it is not seen in patients with a similar degreeof weight loss consequent upon malnutrition.

EXAMPLE 4

Experiments were conducted, to demonstrate that the blockade of the RASis of benefit for cachectic patients, even if previously treated with anACE inhibitor. Patient 1 had cachexia due to chronic heart failure (CHF)(age 74 years, male, weight 50.0 kg, height 178 cm, previous weight loss15.3 kg in 3 years=chronic weight loss). Patient 2 had CHF and a musclemyopathy suffering from idiopathic cachexia (age 38 years, male, weight62 kg, height 180 cm, previous weight loss 11 kg in year=recent weightloss). Each was treated with Losartan (50 mg once daily). Clinicalstatus and parameters of body composition, strength and treadmillexercise capacity were studies, at baseline and during follow-up. Bothpatients had evidence of CHF with impaired exercise capacity andimpaired left ventricular function (LVEF <40%). Both patients had a goodcompliance.

Bioelectrical impedance analysis (patient 1 and 2) was performed in theerect position using a body fat analyser (TANITA TBF-305, TanitaCorporation, IL, USA). Lean and fat mass were automatically analysedbased on equations supplied and programmed into the machine by themanufacturer. These equations are based upon a comparison withmeasurements in a health population.

Dual energy X-ray absorptiometry (DEXA) (patient 1): Whole bodyDEXA-scans were performed using a Lunar model DPXIQ total body scanner(Lunar Radiation Company, Madison, Wis., USA, Lunar system softwareversion 4.3c). The subject was at each time point scanned rectilinearlyfrom head to toe. A scan takes less than 20 min. The mean radiation doseper scan is reported to be about 0.75 μSv (53), about 1/50th of a normalchest X-ray. The DEXA method can be used to obtain from body densityanalyses values of fat tissue mass, lean tissue mass. The technicaldetails of DEXA, performance and segment demarcation have been described(54,55). The error of lean tissue measurements is >2% and of fat tissuemeasurements <5% (56).

Treadmill exercise test (Patients 1 and 2): The patients underwentsymptom limited treadmill exercise testing. A standard Bruce protocolwith the addition of a “stage 0” consisting of 3 min at a speed of 1mile per hour with a 5% gradient was used. The patients breathed througha one-way valve connected to a respiratory mass spectrometer (Amis 2000,Odense, Denmark) and minute ventilation, oxygen consumption and carbondioxide production were calculated on line every 10 seconds using astandard inert gas dilution technique. Patients were encouraged toexercise to exhaustion. Exercise time and oxygen consumption at peakexercise adjusted for total body weight (peak V0₂ in ml/kg/min) weremeasured as an index of the exercise capacity.

Assessment of quadriceps muscle strength (Patients 1 and 2): Thesubjects were seated in a rigid frame, with the legs hanging freely. Aninelastic strap attached the ankle to a pressure transducer. Therecording (Multitrace 2, §, Jersey, Channel Islands) from the pressuretransducer was used to assess strength and to provide visual feedback tothe subject. A plateau of maximum force production indicated that thecontraction was maximal. The best of three voluntary contractions oneach leg, with a rest period of at least one minute in-between, wastaken to represent the maximal voluntary quadriceps muscle strength ofthe right and left leg, respectively.

Results include a follow-up of 126 days for patient 1 and 83 days forpatient 2. Both patients were also studied at intermediate time points.Both patients improved during treatment by 1 NYHA symptom class. In bothpatients, the exercise capacity improved during the study (exercisetime: patient 1 and 2, peak V0₂: patient 2). There was evidence that inboth patients, quadriceps muscle strength improved in both legs. Theseclinical benefits were achieved against the background of a weight gainof 4.6 kg in patient 1 (lean and fat tissue gain), and by stopping theprocess of weight loss and apparently improving the general clinicalstatus and relative muscle performance, i.e. muscle quality (patient 2).No side-effects of treatment were observed.

EXAMPLE 5

The SOLVD treatment study (57) was a randomized, double-blind, andplacebo-controlled trial investigating the effects of enaiapriltreatment in clinically stable patients with a LVEF of 35% or less andevidence of overt congestive heart failure. The precis e details ofstudy organisation, inclusion criteria, run-in period (2 to 7 days) andstabilization period (14 to 17 days), randomisation, treatment titrationand follow-up have been reported previously (57). Based also on data nototherwise available, the results have been re-analysed, restricted tosubjects who participated in the SOLVD treatment trial, who had beenfree of edema at baseline, who had survived for at least 4 monthsthereafter, who had weight measurements at baseline and from at leastone follow-up visit at 4 months or later. The baseline clinicalcharacteristics of these 2082 patients were not significantly differentfrom the characteristics of the total study population.

Of the 2082 patients, 1055 patients were randomised to treatment withenalapril (2.5 to 20 mg per pay) and 1027 patients to treatment withplacebo. Body weight at baseline and during follow-up were measured perprotocol. Body height was not recorded.

Comparison of means between groups was carried out using an unpairedt-test. Comparison of proportions between groups was made by employingthe chi-square test. With regards to the definition of the presence ofcachexia different, a priori suggested, cut-points (58) of 5.0%, 7.5%,10.0% and 15.0% weight loss were considered. To address the question ofwhether or not ACE inhibitors influence the risk of first occurrence ofcachexia, the cumulative incidence of cachexia in the two treatmentgroups was plotted, and analysed employing the log-rank statistic (59).In the analysis of first occurrence of cardiac cachexia, at any givenfollow-up visit, absence of information on cardiac cachexia (i.e. weightnot documented at this visit) is treated as censored. The effect ofcardiac cachexia on survival is assessed using Cox proportional hazardanalysis (58). For these analyses, cardiac cachexia is treated as atime-dependent covariate. The assessment of cardiac cachexia at 4, 8,and 12 months was used in the analysis. These are the time points in thefollow-up period with relatively high proportion of complete informationon cachexia status.

The primary analysis was intention-to-treat. Statistical significance isclaimed at a computed p-value <0.05 (two-sided testing). Estimates ofeffects are provided along with their 95% confidence intervals. Resultsare adjusted for a priori identified prognostic factors such as age,gender, NYHA functional class, LVEF (up to or more than 25%), andtreatment status (enalapril vs placebo, in the case of assessing theeffect of cardiac cachexia on survival).

Of the 2082 CHF patients in this study, 657 (31.6%) developed up to 7.5%weight loss during follow-up. The cumulative frequency of cardiaccachexia increased continuously over time. The frequency of ≧7.5% weightloss (cross-sectional) at 1 year was 8.5% and it increased to 15.5% (2years), and 17.2% (3 years). At baseline, patients who developed cardiaccachexia with ≧7.5% weight loss during follow-up were 1.3 years older(mean 61.2 vs 59.9, p<0.01), had 2.7 kg higher weight (mean 80.5 vs 77.8kg, p<0.001), and they were slightly more frequently treated withdiuretics (87.2 vs 82.6%, p<0.01). Of the patients in this study, 375(18.0%) were female. Female CHF patients developed cardiac cachexia morefrequently (39.5% vs 29.8% in males for ≧7.5% weight loss, p<0.001).Otherwise the baseline clinical characteristics, particularly withregards to NYHA class, LVEF, and disease etiology, of patients whodeveloped cardiac cachexia and those who did not were similar. Thefollowing clinical characteristics at baseline were independentlyrelated to the subsequent development of cardiac cachexia: age (RR ,p<0.00 1), NYHA class, LVEF, and treatment.

The development of cardiac cachexia was closely related to subsequentlyimpaired survival. All a priori identified competitive cut-points forcardiac cachexia were related to impaired survival, independent of theeffects of age, gender, NYHA class, LVEF, and treatment allocation. Ofthe 756 deaths observed during follow-up, 223 occurred in patients whohad been classified as cachectic (≧7.5% weight loss) at the last visitprior to death, i.e. 29.5% of deaths in CHF patients occurred withcardiac cachexia being present. Amongst different cut-offs for cardiaccachexia between 5 and 15%, weight loss 6.5% was the strongest predictorof impaired mortality. The crude effect of cachexia (weight loss ≧6.5%)on survival was highly significant: RR 1.47 (95% confidence interval:1.27 to 1.70), p⁻-⁻0.00000017.

Patients who were allocated to treatment with enalapril had asignificantly lower risk of developing cardiac cachexia duringfollow-up. The crude effect of treatment allocation with enalapril wassignificantly related to a reduced risk of developing cardiac cachexia:RR 0.81 (95% confidence interval: 0.70 to 0.95), p=0.0085. Treatmentallocation to enalapril had a significantly beneficial effect onsurvival independently of the effect of age, gender, NYHA class, andLVEF also in this subset of patients of the SOLVD treatment trial(p<0.01). When adjusted also for the presence of cardiac cachexia (6.5%weight loss) at 4 or 8 months, the treatment effect remainedsignificant. In patients who developed weight loss of at least 7.5% atany time point, only 10 patients with subsequently recorded weightsequal to or higher than the baseline weight were found (enalapril group:6, placebo: 4).

This demonstrates that significant weight loss, i.e. cardiac cachexia,is a frequent event in CHF patients. Weight loss ≧7.5% occurs in about ⅓of patients over 3 years. Spontaneous reversal of the weight loss is avery rare event occurring in less than 2% of cases. Cardiac cachexia isclosely and independently linked to impaired survival of CHF patients.Treatment with an ACE inhibitor, enalapril, in addition to conventionaltherapy, reduced the frequency of the risk of death and the risk ofdeveloping cardiac cachexia. Overall, enalapril therapy reduced the riskof developing cardiac cachexia by 19%.

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1. A method for the treatment of a person, wherein said method comprisesadministering to a person with hypertension and left ventricularhypertrophy in need of such treatment an inhibitor of therenin-angiotensin system wherein the inhibitor is an AT_(I) receptorantagonist comprises losartan in an amount effective to reduce the riskof stroke in patients with hypertension and left ventricularhypertrophy.
 2. A method for the treatment of a person, wherein saidmethod comprises administering to a person 55 years or older at highrisk of developing a major cardiovascular event because of a history ofcoronary artery disease, stroke, peripheral vascular disease, ordiabetes that is accompanied by at least one other cardiovascular riskfactor (hypertension, elevated total cholesterol levels, low HDL levels,cigarette smoking, or documented microalbuminuria), an inhibitor of therenin-angiotensin system, wherein the inhibitor is an AT_(I) receptorantagonist comprises ramipril in an amount effective to reduce the riskof myocardial infarction, stroke, or death from cardiovascular causes.3. The method of claim 2, wherein said method comprises administrationof ramipril in addition to other needed treatment for said risk ofdeveloping a major cardiovascular event.
 4. The method of claim 3,wherein said other needed treatment is selected from the groupconsisting of: antihypertensive medication, anti-platelet aggregatingfactor medication, and lipid-lowering therapy.
 5. A method for thetreatment of a person, wherein said method comprises administering to anadult patient with hypertension and type 2 diabetes mellitus in need ofsuch treatment an inhibitor of the renin-angiotensin system, wherein theinhibitor is an AT_(I) receptor antagonist, wherein said inhibitorcomprises irbesartan in an amount effective for the treatment of renaldisease in said adult patient with hypertension and type 2 diabetesmellitus as part of an antihypertensive medicinal product regimen.
 6. Amethod for the treatment of a person, wherein said method comprisesadministering to a patient with type 2 diabetes and hypertension in needof such treatment, an inhibitor of the renin-angiotensin system, whereinthe inhibitor is an AT_(I) receptor antagonist, wherein said inhibitorcomprises irbesartan in an amount effective for the treatment ofdiabetic nephropathy with an elevated serum creatinine and proteinuria(>300 mg/day) in said patient with type 2 diabetes and hypertension, inan amount sufficient to reduce the progression of nephropathy asmeasured by the occurrence of doubling of serum creatinine or end-stagerenal disease (need for dialysis or renal transplantation).
 7. A methodfor the treatment of a person with stable coronary artery disease with ahistory of myocardial infarction and/or revascularization, wherein saidmethod comprises administering to a person with stable coronary arterydisease in need of such treatment, an inhibitor of the renin-angiotensinsystem, wherein the inhibitor is an AT_(I) receptor antagonist, whereinsaid inhibitor comprises perindopril in an amount effective for thereduction of risk of cardiac events in said patient with a history ofmyocardial infarction and/or revascularization.
 8. A method for thetreatment of a person with stable coronary artery disease, wherein saidmethod comprises administering to a person with stable coronary arterydisease in need of such treatment, an inhibitor of the renin-angiotensinsystem, wherein the inhibitor is an AT_(I) receptor antagonist, whereinsaid inhibitor comprises perindopril in an amount effective to reducethe risk of cardiovascular mortality or nonfatal myocardial infarction.9. The method of claim 1, wherein said method comprises administering toa person with hypertension and left ventricular hypertrophy in need ofsuch treatment an inhibitor of the renin-angiotensin system wherein theinhibitor is an AT_(I) receptor antagonist and wherein said antagonistconsists essentially of losartan in an amount effective to reduce therisk of stroke in patients with hypertension and left ventricularhypertrophy.
 10. (canceled)
 11. The method of claim 9, said losartanprovided as an oral finished pharmaceutical dosage form including atleast one pharmaceutically acceptable excipient.